1 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest H-1085, Hungary.
2 Solvo Biotechnology, Budaörs H-2040, Hungary.
Exp Biol Med (Maywood). 2018 Aug;243(12):1014-1023. doi: 10.1177/1535370218795406. Epub 2018 Aug 15.
MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future. Impact statement Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.
miR-206 是一种非常独特的 miRNA，因为它在横纹肌肉瘤中作为抑制性 miRNA 发挥作用，而在之前的研究中，它可以作为 SMARCB1 免疫阴性软组织肉瘤的致癌 miRNA 发挥作用。本研究旨在研究 miR-206 对其在各种人类肿瘤和正常细胞系中的几个靶基因的影响。在目前的工作中，我们通过永久转染创建了 miR-206 过表达细胞系（HT-1080、Caco2、iASC 和 SS-iASC）。使用定量实时聚合酶链反应、免疫印迹、免疫细胞化学和流式细胞术检测 miR-206 的靶基因（SMARCB1、ACTL6A、CCND1、POLA1、NOTCH3、MET 和 G6PD）和 SMARCB1 蛋白表达。使用 miRNA 抑制来验证我们的结果。我们发现 miR-206 对其靶基因具有不同的沉默作用。虽然观察到总体下调趋势，但个别细胞系的表达谱表现出很大的可变性。只有 CCND1 和 MET 被一致地下调。miR-206 对正常人成纤维细胞系具有抗增殖作用。在 miR-206 转染的 SS-iASC 中，SMARCB1 的强烈沉默效应很可能是由 SS18-SSX1 融合蛋白和 miR-206 的协同影响引起的。在同一细胞系中，通过免疫细胞化学和流式细胞术可以观察到 SMARCB1 蛋白表达的中度降低。在迄今为止对 miR-206 作用的最全面分析中，在所有细胞系中均证实 miR-206 靶基因在 mRNA 水平上的下调程度适中但具有统计学意义。然而，效应的可变性表明该 miRNA 的作用在很大程度上取决于细胞环境。我们的结果还支持这样的概念，即 miR-206 对 SMARCB1 的致癌 miRNA 作用在 SMARCB1 免疫阴性软组织肉瘤中起着重要但不是唯一的作用，因此它可以被认为在未来这些肿瘤的靶向治疗中很重要。
影响说明
miR-206 是一种非常独特的 microRNA，因为它可以根据肿瘤组织作为抑制性 miRNA 或致癌 miRNA 发挥作用。在 SMARCB1 阴性软组织肉瘤中，miR-206 过表达，因此在上皮样和滑膜肉瘤中，它作为致癌 miRNA 发挥作用。miR-206 对其靶基因有不同的沉默作用。我们发现 miR-206 的作用在很大程度上取决于细胞环境。miR-206 在 SMARCB1 阴性软组织肉瘤中的致癌作用至关重要，但并非唯一，在正常人成纤维细胞系中，miR-206 具有抗增殖作用。在肿瘤中观察到的 miR-206 靶基因的表达只能在相应的肿瘤细胞系中重现。这是第一项在正常、肿瘤和基因工程细胞系中研究 miR-206 对其靶基因的永久作用的研究。
